The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing.
The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing.
复制标题
DOI：
10.1371/journal.ppat.1007315
复制
发表时间：
2018-09
期刊：
影响因子：
6.7
通讯作者：
Clayton C
中科院分区：
文献类型：
--
作者：
Begolo D;Vincent IM;Giordani F;Pöhner I;Witty MJ;Rowan TG;Bengaly Z;Gillingwater K;Freund Y;Wade RC;Barrett MP;Clayton C
动质体寄生虫锥虫和利什曼原虫感染了数百万人，并导致经济上毁灭性的牲畜疾病，现有的少数药物存在严重缺陷。基于苯并氧杂硼杂环戊烯的化合物是非常有前途的潜在新型抗锥虫疗法，候选物已经在人类和动物临床试验中。我们研究了几种苯并氧杂硼杂环戊烯的作用机制，包括AN 7973，一种早期的兽医锥虫病候选药物。在所有动质体中，转录是多顺反子的。单个mRNA的5 '端是通过短前导序列的反式剪接以及前一个mRNA的偶联聚腺苷酸化产生的。用AN 7973处理布氏锥虫在1小时内抑制反式剪接，如通过Y结构剪接中间体的损失、mRNA水平降低和核周颗粒的积累所判断的。剪接的前导前体RNA的甲基化不受影响，但更长时间的AN 7973处理引起S-腺苷甲硫氨酸和甲基化赖氨酸的增加。总之，结果表明mRNA加工是AN 7973的主要靶点。多聚腺苷酸化是动质体反式剪接所必需的，AN 7973在T.过表达T.布氏裂解和多聚腺苷酸化因子CPSF 3，确定CPSF 3为潜在的分子靶点。分子模拟结果表明，AN 7973抑制CPSF 3是可行的。因此，我们的研究结果在化学上验证了mRNA加工作为锥虫中可行的药物靶点。其他几种苯并氧杂硼杂环戊烯显示出与AN 7973相似的代谢组学和剪接效应，将剪接抑制确定为常见的作用模式，并表明其可能与甲基化代谢物的后续变化相关。然而，CPSF 3表达后的颗粒形成、剪接抑制和抗性并不总是相关的，并且AN 7973中锥虫的延长选择仅导致1.5倍的抗性。因此，靶向锥虫mRNA加工的氧杂硼杂环戊烯的作用模式可能超出CPSF 3抑制。锥虫和利什曼原虫感染了数百万人，并导致经济上毁灭性的牲畜疾病;现有的少数药物存在严重缺陷。由刚果锥虫（Trypanosoma congolense）和间日锥虫（Trypanosoma vivax）引起的牛锥虫病在非洲是一个严重的问题，因为牛不仅用于食物，而且用于牵引，需要新药。单次注射苯并氧杂硼杂环戊烯化合物AN 7973固化T.牛和山羊的刚果人感染。虽然对T.尽管间日疟原虫排除了AN 7973作为商业上可行的治疗牛锥虫病的药物的开发，但它仍然可能具有由其他锥虫引起的疾病的潜力。我们使用了大量的方法来找出AN 7973如何杀死锥虫，并将其与其他苯并氧杂硼杂环戊烯进行了比较。AN 7973和其他一些化合物对寄生虫代谢的影响类似于以前对正在测试人类昏睡病的苯并氧杂硼杂环戊烯的影响。然而，AN 7973的最快速作用是对锥虫mRNA的加工。结果，mRNA的量减少，蛋白质的合成停止。我们得出结论，AN 7973和其他一些苯并氧杂硼杂环戊烯通过停止基因表达杀死锥虫。
Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition. Trypanosomes and leishmanias infect millions of humans and cause economically devastating diseases of livestock; the few existing drugs have serious deficiencies. Trypanosomosis of cattle caused by Trypanosoma congolense and Trypanosoma vivax, is a serious problem in Africa because cattle are used not only for food but also for traction, and new drugs are needed. A single injection of the benzoxaborole compound AN7973 cured T. congolense infection in cattle and goats. Although slightly lower effectiveness against T. vivax precludes development of AN7973 as a commercially viable treatment against cattle trypanosomosis, it could still have potential for diseases caused by other trypanosomes. We used a large range of methods to find out how AN7973 kills trypanosomes, and compared it with other benzoxaboroles. AN7973 and some of the other compounds had effects on parasite metabolism that resembled those previously seen for a benzoxaborole that is being tested for human sleeping sickness. The most rapid effect of AN7973, however, was on processing of trypanosome mRNA. As a consequence, amounts of mRNA decreased and synthesis of proteins stopped. We conclude that AN7973 and some other benzoxaboroles kill trypanosomes by stopping gene expression.
登录
查看更多内容
影响因子：
2.7
作者：
Akama T;Zhang YK;Freund YR;Berry P;Lee J;Easom EE;Jacobs RT;Plattner JJ;Witty MJ;Peter R;Rowan TG;Gillingwater K;Brun R;Nare B;Mercer L;Xu M;Wang J;Liang H
通讯作者：
Liang H
发现新型基于苯并恶硼罗的有效抗锥虫药物。
影响因子：
4.2
作者：
Ding, Dazhong;Zhao, Yaxue;Plattner, Jacob J.
通讯作者：
Plattner, Jacob J.
影响因子：
4.4
作者：
Antwi EB;Haanstra JR;Ramasamy G;Jensen B;Droll D;Rojas F;Minia I;Terrao M;Mercé C;Matthews K;Myler PJ;Parsons M;Clayton C
通讯作者：
Clayton C
影响因子：
2.6
作者：
Eisler, MC;Brandt, J;Geerts, S
通讯作者：
Geerts, S
影响因子：
2.7
作者：
Akama, Tsutomu;Baker, Stephen J.;Plattner, Jacob J.
通讯作者：
Plattner, Jacob J.